Reverse transcriptases (RTs) frequently switch templates, leading to an increase in genetic variation and retroviral recombination. We have performed a series of studies in which high-frequency deletion of homologous repeats was used as a model system for probing the mechanism of RT template switching and in vivo reverse transcription. We have determined the effects of the size of the repeats, distance between repeats, RNA secondary structure, intracellular nucleotide pools, and mutations in murine leukemia virus (MLV) RT on the frequency and locations of RT template switching. These studies led to the proposal of a new dynamic copy-choice model for retroviral recombination, which postulated that there is a steady state between the rates of DNA synthesis and RNA degradation and that conditions affecting this steady state influence the frequency of RT template switching. Analysis of direct-repeat deletions has also led to new insights into the in vivo mechanism of reverse transcription. The results of these studies have provided the first demonstration of polymerase-dependent ribonuclease H (RNase H) activity in vivo and indicated that MLV nucleocapsid (NC) protein increases the in vivo rate of DNA synthesis, especially in regions of the template containing secondary structures. We plan to determine the roles of HIV-1 RT, NC, and RNA secondary structure in RT template switching. To gain insights into the mechanisms of in vivo reverse transcription, we are analyzing the role of MLV and HIV-1 NC zinc fingers and basic residues in minus-strand and plus-strand transfer. We are also analyzing the effects of RNA secondary structure on the rate of reverse transcription. Finally, we are investigating whether MLV- and HIV-1-based vectors containing two primer-binding site (PBS) regions can initiate reverse transcription more than once; we are also using this system as a sensitive in vivo assay to analyze the effects of cis-mutations in the PBS regions on the efficiency of DNA synthesis initiation. Corresponds to Pathak Project 1 in the May 2003 site visit report of the HIV Drug Resistance Program